DNA Repair

The Rev1 deoxycytidyl transferase functions as a scaffold protein for DNA polymerase {zeta} (Pol {zeta})-mediated translesion synthesis (TLS). Biochemical studies with yeast enzymes indicate that Rev1 plays a dual regulatory role in TLS, stimulating Pol {zeta} activity at sites of damage but inhibiting its activity on undamaged DNA. An evolutionary conserved N-terminal alpha-helical motif (M1), located 10-20 amino acids upstream of Rev1s single BRCT domain, is required for the inhibitory activity of Rev1 on undamaged DNA. Mutations in the M1 motif result in a stimulation of Pol {zeta} replication activity on both undamaged and damaged DNA. Yeast cells carrying a REV1 mutant lacking the M1 motif, show a significant increase in mutation track length, without significantly affecting overall spontaneous mutation rates. The regulatory activity of Rev1 is independent of its catalytic activity. However, it requires that Rev1-Pol {zeta} is a stable complex, and that this complex is coordinated by the replication clamp PCNA. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/700666v1_ufig1.gif" ALT="Figure 1"> View larger version (10K): org.highwire.dtl.DTLVardef@9b40dorg.highwire.dtl.DTLVardef@10bf5d1org.highwire.dtl.DTLVardef@376ff4org.highwire.dtl.DTLVardef@1970db8_HPS_FORMAT_FIGEXP M_FIG C_FIG

RPA is a heterotrimeric ssDNA binding protein that is highly conserved across all eukaryotes. Arabidopsis (Arabidopsis thaliana) has five RPA1 paralogs divided into three groups (A, B, C) each with unique functions in DNA replication and repair. The group C paralogs (RPA1C and RPA1E in Arabidopsis) function specifically in DNA-damage repair and carry a C-terminal extension unique to group-C paralogs. This C-terminal extension contains a zinc finger motif (ZFM) that is highly conserved and is therefore predicted to be critical to the functionality of the paralogs during DNA damage repair. To address this, we employed a CRISPR-Cas9 strategy to specifically remove the ZFM from RPA1C or RPA1E while leaving the genes otherwise intact (termed C-ZFKO and E-ZFKO). C-ZFKO and E-ZFKO lines were challenged with DNA damaging agents, and their susceptibility was compared to both WT (Col-0) lines and to previously characterized T-DNA null mutants (rpa1c and rpa1e). To address the role of the respective ZFMs in homologous recombination pathways (HRR), we employed a GUS-reporter system to compare WT lines to C-ZFKO and E-ZFKO lines. We find here that C-ZFKO and E-ZFKO lines displayed hypersensitivity to DNA damaging agents at a level comparable to previously characterized T-DNA null mutants (rpa1c and rpa1e). When studying the rate of HRR, both C-ZFKO and rpa1c showed a drastic reduction in single-strand annealing (SSA) while E-ZFKO and rpa1e had a more modest, but still significant decrease. All mutant lines had a comparable decrease in synthesis-dependent strand annealing (SDSA) compared to WT. Thus, we show here that the respective RPA1C and RPA1E-encoded ZFM is crucial for the ability of each paralog to function during DNA damage repair.

Caffeine, the most widely consumed stimulant worldwide and primarily sourced from coffee, is well known for its central nervous system effects. Emerging evidence indicates that caffeine also modulates key cellular processes, including DNA repair. It inhibits the kinase activity of ATM and ATR-essential DNA damage response proteins, and impairs homologous recombination (HR)-mediated repair through multiple mechanisms. However, its effects on nonhomologous end joining (NHEJ), a major double-strand break (DSB) repair pathway, have been underexplored. In a recent study, we reported that caffeine inhibits NHEJ primarily by interfering with Ligase IV/XRCC4 complex, using in vitro and ex vivo model systems. Given coffees role as a primary dietary caffeine source, this study investigates the impact of Coffea arabica decoction on NHEJ-mediated DSB repair. High-performance liquid chromatography (HPLC) quantified caffeine levels in the decoction, followed by in vitro and ex vivo assays to evaluate NHEJ efficiency. Results demonstrate that coffee decoction inhibits end joining of both compatible and noncompatible DNA ends in cell-free systems derived from normal and cancer cells. Extrachromosomal repair assays confirmed impaired intracellular NHEJ, leading to accumulation of unrepaired DSBs in human cells. Kinetic analysis of {gamma}-H2AX foci formation and resolution revealed persistent DNA breaks and reduced repair kinetics. Reconstitution experiments verified that the decoction specifically targets the Ligase IV/XRCC4 complex. These findings, building on our previous work, establish coffee decoction as a potent NHEJ inhibitor, mirroring purified caffeines effects. This underscores caffeines interference with endogenous DNA repair, with profound implications for cancer therapy by sensitizing tumors to genotoxic treatments.

In many eukaryotic species, DNA is methylated at the 5 position of cytosine to form 5mC, predominantly within CG dinucleotides. Despite being conserved since the dawn of eukaryotic life, 5mC is often lost from individual lineages, suggesting that it may have detrimental effects. One such effect is genotoxicity, through the effect of 5mC on the process of cytosine deamination and its repair. Additionally, enzymes that introduce 5mC (DNA methyltransferases, DNMTs) can also damage DNA through alkylation and oxidative stress, but how these genotoxic effects combine to influence mutagenesis is unclear. To investigate how mutagenesis changes upon methylation of CG dinucleotides we introduced high levels of CG methylation into the bacteria E. coli. 5mC induction increased mutation at CG dinucleotides consistent with increased C to T mutations. We also discovered that 5mC induction led to increased mutations at AT base pairs, specifically in the absence of the alkylation repair enzyme AlkB. This effect was specific to certain E. coli strains and was not dependent on the DNA repair enzyme RecA, so its exact mechanism remains unclear. Together, our work highlights multiple mutagenic consequences of DNMT expression, which might act as selective pressures for organisms to lose 5mC across evolution.

Allele conversion describes a process where a heterozygous variant is made homozygous. Recently, it has been shown that allele conversion can be triggered by DNA damage at the heterozygous site. This process has the potential to repair pathogenic heterozygous mutations; however, the efficiency is low. Here, we endeavoured to understand the mechanism underlying allele conversion, ultimately to raise allele conversion efficiency to functionally relevant levels. To test this, we developed a Compound Heterozygous Allele Conversion Reporter (CHACR) cell line. This line comprises knocked-in fluorescent protein encoding genes, with heterozygous inactivating mutations resulting in different fluorescence profiles from each allele. These mutations create protospacer adjacent motifs (PAM) for Cas9 recognition, where allele-specific gRNAs (AS-gRNAs) target the heterozygous mutations. We showed that applying these AS-gRNAs with either Cas9 nuclease or Cas9(D10A) nickase can recover mCherry fluorescence. Sorting and sequencing these fluorescent cells revealed wild-type sequences, suggesting allele conversion repaired the mutation using the homologous allele as a template. Allele conversion can also be triggered using an adenine base editor with an AS-gRNA, and this allele conversion mechanism can be manipulated by inhibiting DNA-PKcs or overexpressing RAD51. This work introduces a model for measuring allele conversion, and modifiers of this mechanism.

Latent HIV-1 proviruses remain the major barrier to curing HIV infection. Although many of these proviruses are defective, with large internal deletions and hypermutations, the mechanisms underlying their formation are still poorly understood. In this study, we applied CRISPR/Cas9 knockout screens to identify DNA damage response (DDR) proteins that contribute to the formation of defective HIV-1 proviruses carrying large internal deletions. Using an HIV-1-based dual-fluorophore vector as a model, we distinguished cells harbouring intact proviruses from those carrying large internal deletions by flow cytometry and cell sorting. We then validated top candidates using CRISPR-mediated gene activation and small interfering RNA-mediated knockdown, and we measured gene and protein expression by quantitative PCR and Western blotting. Across these approaches, the helicase-like transcription factor HLTF emerged as a consistent modulator of large internal deletions: increased HLTF expression raised the proportion of cells carrying defective proviruses, whereas reduced HLTF expression had the opposite effect. Additional repair factors, including RAD1, RAD18, TREX2, and ZRANB3, also influenced the balance between intact and defective proviruses, suggesting that multiple DNA repair pathways cooperate in this process. Deep sequencing of reporter proviruses confirmed the presence of large internal deletions in the populations identified as defective. Our data indicate that several DNA damage response proteins, including HLTF, are involved in the generation of defective proviruses and may constitute a previously undescribed host defense mechanism against HIV-1. Authors SummaryWhen HIV-1 infects a cell, it copies its genetic material (RNA) into DNA and inserts this DNA into the cells genome, giving rise to proviruses that can persist for long periods and become part of the host DNA. Many of these viral DNA copies are defective, often missing large parts of their genome, but we still do not fully understand how these large deletions arise. In this study, we used a genetic screening approach to switch off many human DNA repair genes and asked how this affected the balance between intact and defective HIV proviral DNA. We used an HIV-1-based dual-colour reporter vector allowing us to distinguish intact from deleted viral DNA by simple fluorescence read-outs. We found that several human DNA repair factors, in particular a protein called HLTF, change how often large deletions appear. Our results suggest that normal DNA repair processes in infected cells can sometimes turn incoming HIV-1 DNA into defective forms that cannot support productive infection. This work points to host DNA repair as a contributor to the large pool of defective HIV-1 DNA seen in people with HIV (PWH) and raises the possibility that these pathways could one day be harnessed to make infections less harmful.

Sirtuin 6 (SIRT6) is an important regulator of DNA repair, metabolism, chromatin maintenance and longevity. SIRT6 Serine 10 phosphorylation controls SIRT6 recruitment to the sites of DNA damage. To explore the effect of SIRT6 Serine 10 phosphorylation on lifespan, we generated two SIRT6 mutant mouse strains: phospho-null S10A and phosphomimetic S10E. The S10E mutant mice demonstrated enhanced DNA repair capacity, elevated LINE1 expression and reduced lifespan in male mice compared to the wild-type and S10A mice. This result suggests that SIRT6 S10E mutation enhances DNA repair capacity at the expense of reduced LINE1 silencing leading to shorter lifespan. While both SIRT6 functions in DNA repair and chromatin maintenance are important for longevity, our results suggest that when the balance between these functions is shifted, diminished of LINE1 control has a stronger impact on lifespan than enhanced DNA repair.

The MYC associated factor X (MAX) is the heterodimeric partner of the MYC paralogs (MYC, MYCN and MYCL). When deregulated, high level of the MYC paralogs contribute to all aspects of tumorigenesis and tumor growth. MAX can also heterodimerize with the MXD proteins, MNT and MGA. Heterodimerization and sequence specific DNA binding to the E-Box sequences at gene promoters is controlled by their heterodimerization with the MAX b-HLH-LZ. As a heterodimer with MAX, MYC proteins activate genes involved in cell metabolism, growth and proliferation whereas MXD proteins, MNT and MGA repress them. MAX can also bind to the E-Bos sequence as a homodimer. Being devoid of a transactivation domain it can act as an antagonist of the MYC/MAX heterodimers. Variants of MAX have been reported to be linked to cancer. These variants are either not expressed, inactivated or lead to missense mutations. This has led to the notion that MAX may have a tumor suppressor role. Here, we characterize three of those variants with missense mutations in the basic region, i.e. E32K, R35P and R35C. We analyzed their heterodimerization with the b-HLH-LZ of MYC and their DNA binding properties as homo-and heterodimers. The R35C variant b-HLH-LZ was found to have a markedly increased affinity for the b-HLH-LZ of MYC. We also observed that all three b-HLH-LZ variants have a lower affinity as homodimers for the E-Box than the WT. This was shown to lead to a preferential binding of all the heterodimeric b-LHLH-LZ to the E-Box. This effect is exacerbated in the case of the R35C variant. We argue that this preferential binding of MYC as heterodimers with these variants to E-Box sequences could contribute to tumorigenesis. Hence, our results suggest that, mechanistically, the MAX homodimer bound to the E-Box could act as a tumor suppressor. MATERIALS AND METHODSO_ST_ABSMolecular modelingC_ST_ABSThe open source version 1.7.6.0 of Pymol was used for modeling and molecular rendering [1]. The crystal structure of the MAX homodimer bound to the E-Box (1HLO [2]) was used as a template for the generation of the models. The variants were generated using the mutagenesis function in the wizard. The conformation of the K32 side chain was manually set in order to avoid introducing steric clashes with DNA. Protein expression and purificationThe cDNA, coding for the MAX b-HLH-LZ (Max* hereafter, residues 22-103, UniProt entry P61244-1) to which are added the GSGC residues in c-terminal, inserted in the pET3a vector was already available in the laboratory [3] and was used as a template to generate the plasmids with inserts coding for each of the mutants (E32K, R35C and R35P) through quick-change PCR with Q5 DNA polymerase and DpnI from New England Biolabs. The primers used were purchased from IDT DNA, their sequences are listed in Table S1. Sequence for each construct was confirmed by Sanger sequencing at the Plateforme de sequencage SANGER - Centre de recherche du CHU de Quebec - Universite Laval. The primary structure for the basic region of each construct is given in Fig. 2A. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/715400v1_fig2.gif" ALT="Figure 2"> View larger version (41K): org.highwire.dtl.DTLVardef@1b05d5eorg.highwire.dtl.DTLVardef@1c1d692org.highwire.dtl.DTLVardef@ee469dorg.highwire.dtl.DTLVardef@15e0ba4_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 2.C_FLOATNO Structure schematics, specific and non-specific interactions dictating specificity and stability of binding of the basic region of MAX to the canonical (CACGTG) E-Box. A. Primary structure for the basic region of MAX and each of the variants. Positions making the most important contacts with the E-box are indicated by black arrows. Positions for the variants studied here are colored according to the Zappo colour scheme, following their physico-chemical properties: red for negative, blue for positive, magenta for proline and yellow for cysteine. B. The side chain (carboxylate) of E32 receives H-Bonds from the CA nucleobases in the leading strand (white carbon atoms). R35 and R36 make a salt bridges with phosphate groups while and the guanidino moiety of R36 makes a specific H-Bond with the nucleobase of the G in the strand of the reverse complement (cyan carbon atoms). C. The R35C mutation removes one non-specific salt-bridge at the interface of the complex. D. The aliphatic portion of the K side chain in the E32K variant is unable to accept the H-Bonds from the CA nucleobases and leads to the stabilisation of the complex and the helical structure of the basic region. E. In addition to removing a salt-bride, the Pro residue in the R35P kinks the path of the basic region, prevents the establishment of the specific H-Bonds mandatory for recognition of the E-Box and leads to unfolding of the helical state. C_FIG The MYC b-HLH-LZ (Myc*), the Max*WT b-HLH-LZ and its variants were expressed and purified as previously described [3,4] After lyophilisation, the b-HLH-LZs were kept at -20{degrees}C and solubilised in Myc buffer (50 mM NaCl, 50 mM NaH2PO4 pH 5.5) for Myc* or PBS for Max* at a final concentration of 1 mM before use. Circular dichroismAll circular dichroism (CD) measurements were performed on a Jasco J-810 spectropolarimeter equipped with a Peltier-type thermostat. The instrument was routinely calibrated using an aqueous solution of d-10-(+)-camphorsulfonic acid at 290.5 nm. Samples were prepared as follows: Max* (either WT or a variant) was diluted in 100 {micro}l 2X CD buffer (40 mM KCl, 11.4 mM K2HPO4, 28.6 mM KH2PO4, pH 6.8) and the volume adjusted to 106 {micro}l with PBS. 10 {micro}l TCEP 16 mM were added, and the volume further adjusted to 192 {micro}l with ddH2O before samples were incubated overnight at room temperature. After reduction, Myc* was added and the volume adjusted to 198 {micro}l with Myc buffer (Na2HPO4 0.95 mM, NaH2PO4 49.05 mM, 50 mM NaCl, pH 5.5). The DNA complexes were prepared as follows. After a 10 minutes incubation of the protein samples at room temperature, 0, 1 or 2 {micro}l of 2 mM of specific or non-specific DNA duplexes in 10 mM Tris pH 8.0 were added and the volume adjusted to 200 {micro}l with 10 mM Tris pH 8.0. The strands of the specific probe were: 5-ATT ACC CAC GTG TCC T*AC-3 and 5-GTA GGA CAC GTG GGT* AAT-3 (with the E-box sequence underlined) and the non-specific probe: 5-ATT ACC TCC GGA TCC T*AC-3 and 5-GTA GGA TCC GGA GGT* AAT-3 (Integrated DNA Technologies). Samples were further incubated for 10 minutes at room temperature and transferred to a 1 mm path length quartz cuvette. All spectra were recorded from 250 to 195 nm at 0.1 nm intervals by accumulating 10 spectra at 25 {degrees}C. Thermal denaturations were recorded at 222 nm from 5 to 95 {degrees}C at a heating rate of 1 {degrees}C/min. CD signal for spectra and thermal denaturations was corrected by substracting the signal from corresponding spectra or thermal denaturation either for buffer alone or the appropriate DNA duplex. CD signal was then converted to mean residue ellipticity using the following formula [5]: [{theta}] = {delta} {middle dot} MRW/(10{middle dot}c l) where [{theta}] is the mean residue ellipticity in deg {middle dot} cm2 dmol-1, {delta} is the CD signal in millidegrees, MRW is the mean residue weight, c is the concentration in mg/ml and l is the pathlength in mm. For the heterodimers, the concentration used was the sum of Max* and Myc* and the MRW was determined using a weighted average.

The base excision repair (BER) glycosylase MUTYH initiates repair of 8-oxo-7,8-dihydroguanine (OG): adenine (A) mispairs to prevent G to T transversion mutations. Inherited biallelic mutations in MUTYH are correlated with the cancer pre-disposition syndrome MUTYH-associated polyposis (MAP) and contribute to an increased lifetime risk of colorectal cancer. Over 1,000 germline and somatic MUTYH variants have been reported that are associated with MAP and other cancers, but for most the functional impact is unknown. Herein, we examined a subset of cancer-associated variants (CAVs) localized in the interdomain connector (IDC), which links the N-terminal adenine excision and C-terminal OG recognition domains via its zinc linchpin motif and serves as a hub for downstream repair interactions. In vitro assays measuring glycosylase activity, lesion affinity, and AP endonuclease stimulation revealed no substantial defects relative to wild-type MUTYH. In contrast, a newly optimized mammalian cell assay revealed some IDC variants exhibit reduced repair. These results suggest that some variants disrupt steps downstream of adenine excision, whereas others impair lesion recognition and base excision. This work underscores the value of independent functional assays for accurately assessing variant dysfunction and classification. Analysis of MUTYH variants highlights the complexity of the roles of MUTYH in preserving genomic integrity.

BackgroundQN-302 is a tetra-substituted naphthalene diimide (NDI) compound designed to interact with G-quadruplex (G4) DNA. QN-302 is currently being evaluated in a phase 1 clinical trial on patients with advanced pancreatic ductal adenocarcinoma (PDAC) and other solid tumors. However, the mechanistic origin(s) of its anticancer activity remains to be fully understood. ResultsWe report herein the ability of QN-302 to damage DNA at G4 sites in cancer cells. To this end, we implemented a series of in vitro assays (FQA and FRET-melting) and cell-based techniques (in situ click imaging and immunodetection) that concurred in demonstrating both the DNA damaging properties of QN-302 and its ability to engage G4s in human cancer cells. Then, we investigate its anticancer effects in PDAC (MIA PaCa-2 cells) and show that it can be efficiently potentiated upon combination with Olaparib, an inhibitor of DNA repair, in an approach referred to as chemically induced synthetic lethality. ConclusionThis study not only confirms the excellent anticancer properties of QN-302 in human cancer cells but also provides insights into its mechanism of action. The optimization of this therapeutic activity by combination with Olaparib opens a promising new avenue for improving its clinical efficacy.

AO_SCPLOWBSTRACTC_SCPLOWProtein filaments play fundamental functions in the cell, ranging from scaffolding like in the cytoskeleton to sensing and transmitting forces and torques. Here we address the case of the nucleoprotein filaments (NPFs) of homologous recombination (HR) formed by the polymerization of the RecA protein on DNA. In contrast to the cytoskeleton filaments, the HR filaments are not known to exert or sense forces. However the stress in the stretched and unwound DNA bound to those filaments was shown to play a role in promoting DNA strand exchange during the early stage of the HR mechanism. Here we use molecular dynamics simulations to examine whether the strain in the nucleoprotein filament upon strand exchange progression and D-loop formation may influence subsequent steps of the HR process. Our results indicate that the filament mechanical properties are sensitive to the length of DNA incorporated in the D-loop. The response we observe upon increasing the D-loop length is first elastic, up to a threshold that we estimate to be 27 incorporated base pairs, after which the NPF enters a plastic stage where the protein-DNA connectivities are reorganized. Notably, the DNA displaced strand locally switches from site II to site III, a newly characterized binding site. We discuss the possible consequence of this mechanical response of the NPFs for the course of the HR process.

Telomeres are nucleoprotein structures at the ends of eukaryotic chromosomes that safeguard them from triggering inappropriate DNA damage signaling. POT1, a member of the mammalian shelterin complex, binds single-stranded (ss) telomeric DNA and blocks the activation of the ATR kinase-mediated DNA damage response at telomeres. Yet until recently, it was poorly understood how the double-stranded (ds)-ss telomeric junction was protected from DNA damage response factors. An initial study of the DNA-binding activity of human POT1 (hPOT1) using systematic evolution of ligands by exponential enrichment (SELEX) and subsequent investigation revealed that POT1 contains a binding pocket, known as the POT-hole, that binds the 5 phosphorylated dC of the telomeric ds-ss junction. The amino acid residues composing the POT-hole show full sequence identity with telomeric proteins from diverse eukaryotes, including Caenorhabditis elegans POT-1. The current study builds on this SELEX method, developing an extensive analysis pipeline for SELEX datasets sequenced by next-generation sequencing and achieving a deeper analysis of the resulting sequences. We validated our approach by applying it to the DNA-binding domain of hPOT1, yielding results consistent with a previous SELEX study. Furthermore, we employ our pipeline to characterize the DNA-binding activity of C. elegans proteins that are considered homologs of hPOT1: POT-1, POT-2, POT-3, and MRT-1. Our analysis suggests that all four proteins show a binding preference for G-enriched DNA sequences, with POT-1 additionally binding secondary structural elements. Overall, we present a bioinformatics pipeline that is accessible and applicable for determining the nucleic acid-binding properties of a variety of proteins.

DNA-binding proteins must quickly locate specific sites on DNA to enable replication, repair, and transcription. While sequence-specific recognition is well understood, the physical basis of structure-specific recognition remains unclear, limiting our understanding of DNA damage repair. Proteins must distinguish damaged sites within largely undamaged DNA; however, studying this is challenging due to DNAs dynamic nature. We hypothesised that DNA damage causes changes in DNA structure, signalling protein recruitment. Using confocal single-molecule FRET, we analysed seven DNA duplexes containing modifications such as ribonucleotide, 8-oxoguanine (8-oxoG), abasic sites, nicks, and gaps, which are all involved in the base excision repair (BER) pathway. Each construct was measured with nine dye pairs in triplicate to capture changes in bending, twisting, and stretching. An automated analysis pipeline processed 162 measurements, enabling rigorous statistical comparisons. All modifications altered FRET efficiencies compared to undamaged DNA, including the subtlest change: a single oxygen difference (ribo-vs deoxyribonucleotide). Abasic sites, nicks, and gaps had the greatest effects. These findings provide direct evidence that DNA damage affects duplex structure and dynamics beyond the lesion site, suggesting DNA flexibility changes may act as an early signal for repair protein recruitment. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/709887v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@a85839org.highwire.dtl.DTLVardef@3813dborg.highwire.dtl.DTLVardef@19fa06aorg.highwire.dtl.DTLVardef@dc9729_HPS_FORMAT_FIGEXP M_FIG C_FIG

MutY excises adenine (A) from 8-oxo-guanine:adenine (OG:A) lesions in DNA to initiate base excision repair (BER) and thereby prevent mutations. A catalytic Glu, found at position 43 in the enzyme from Geobacillus stearothermophilus (Gs MutY), protonates the nucleobase at N7 to labilize the N-glycosidic bond. The resulting oxocarbenium ion transition state is stabilized by a covalent DNA-enzyme intermediate and resolved by nucleophilic attack to yield the beta-anomer abasic AP site product. The retaining SN1 mechanism for MutY posits deprotonation of the nucleophile by the catalytic Glu. Here we tested kinetic and structural consequences of Glu replacement and found that E43Q and E43S substitution variants were severely impaired, retained measurable activity, but engage the substrate nucleobase in an anti conformation, rotated by 180 {degrees} from the syn conformation seen in previous substrate complexes. The enzyme-generated AP product is observed in its alpha-anomer configuration for these Glu-replacement variants. Comparison with inverting adenine glycosylases that act on RNA or nucleosides shows that MutYs mechanism is uniquely reliant on one catalytic residue for both leaving group and nucleophile activation, a situation that may serve to ensure only rare adenines paired with OG are excised.

One challenge to DNA replication is the presence of unrepaired damage on the template strand, which can stall the replication machinery. This stall can be resolved by the translesion synthesis (TLS) pathway, in which specialized translesion polymerases are recruited to copy damaged DNA. Because TLS polymerases are error-prone, their activity is regulated at multiple levels to minimize unnecessary mutagenesis. Although the molecular mechanisms of bacterial TLS have been extensively studied in Escherichia coli, less is known about this pathway in other species. In E. coli, the TLS polymerase Pol IV is minimally enriched at replication forks in the absence of DNA damage but is strongly recruited upon replication stalling, enabling TLS while minimizing mutagenesis. However, we recently showed that the Bacillus subtilis TLS polymerase Pol Y1, the homolog of Pol IV, is moderately enriched near replication sites even during normal growth and is not further enriched upon treatment with the DNA damaging agent 4-nitroquinoline 1-oxide (4-NQO). It is unknown whether this behavior is unique to 4-NQO or general to other types of DNA damage. In this study, we investigate the effects of four different DNA damaging agents (ultraviolet light, methyl methanesulfonate, nitrofurazone, and mitomycin C) in B. subtilis. We first characterize the contributions of the two TLS polymerases, Pol Y1 and Pol Y2, to DNA damage survival and damage-induced mutagenesis after treatment with these agents. We then use single-molecule fluorescence microscopy to measure the localization and dynamics of individual Pol Y1 molecules in live B. subtilis cells. We find that Pol Y1 and Pol Y2 have differing effects on survival and mutagenesis, but that under no circumstances is Pol Y1 strongly recruited to sites of replication upon DNA damage. This study broadens our understanding of TLS in B. subtilis, indicating that there are notable differences in TLS mechanisms across bacteria.

Long non-coding RNAs (lncRNAs) account for a major proportion of the transcriptional output in complex organismal genomes. Their emergence as auxiliary regulators of gene expression as well as their roles in metastasis and cancer progression has put them in the limelight. LncRNAs perform multitudes of functions and often moonlight as regulators, scaffolds and guides. Most lncRNAs are cell and tissue specific and can act as markers for diseases as well as targets for therapeutic interventions. LncRNAs are also known to make use of higher order structures such as G-quadruplexes (G4) to facilitate complex functions and interactions. THAP9-antisense1 (AS1) is a lncRNA coding gene (recently annotated by Ensembl) that codes for 12 lncRNA transcripts and has been implicated in many disease pathologies like gastric cancer, spontaneous neutrophil apoptosis, hepatocellular carcinoma, and the progression of oesophageal cancer. It is the antisense gene pair of the THAP9 gene ( a transposase derived gene) with which it shares a promoter. THAP9-AS1 has been reported to be dysregulated during stress and several cancers. However, the exact role of the lncRNA is not well understood. Bioinformatics driven strategies are used to identify putative quadruplex forming sequences (PQSs) within the lncRNA THAP9-AS1. The identified PQSs are further validated using biophysical, spectroscopic and molecular biology driven techniques. The importance of each G-tract in the formation of a particular RNA G-quadruplex (rG4) is studied via the investigation of several deletion mutants. The findings demonstrate the rG4 forming potential of the identified PQSs within THAP9-AS1.

AO_SCPLOWBSTRACTC_SCPLOWThe cellular transcription factor BCL11b (B-cell CLL/lymphoma 11b) interacts with numerous cellular and viral factors to modulate gene expression positively or negatively. Post-translational modifications of BCL11b, such as SUMOylation and phosphorylation, have been documented to switch its transcriptional activity from a repressor to an activator state. In the present study, we investigated the acetylation of BCL11b and we identified the histone acetyltransferase p300 as able to acetylate BCL11b. Subsequently, we observed that the mutation of the lysine K686 residue of BCL11b (BCL11b K686R) influenced its global acetylation. Furthermore, the BCL11b K686R mutation also modulated the transcriptional regulation of BCL11b, including its activity in regulating the p21 and IL-2 promoters. This effect on transcriptional regulation was due to the importance of the lysine K686 residue for BCL11b nuclear localization. Our results underscore the critical role of the lysine K686 residue in BCL11b for its interaction with p300 and its nuclear localization, suggesting a possible function of p300 in the nuclear transport of BCL11b. Collectively, our findings contribute to a better understanding of BCL11b-mediated gene expression and of the interactions of BCL11b with cellular partners.

Escherichia coli YicC enzyme is the founding member of a family of endoribonucleases that is encoded in virtually all bacterial species. Previous structural studies revealed that this ribonuclease binds RNA by a novel mechanism in which the hexameric apoprotein presents an open channel that undergoes a large rotation upon RNA binding and clamps down on the RNA. The current study follows up on these findings by examining the cleavage of various oligonucleotide substrates designed to probe recognition elements required for YicC binding and cleavage. A 26-nucleotide RNA oligomer (oligo), with a KD in the low micromolar range, was the standard to which numerous oligos with altered sequence were compared. In vitro RNase assays and fluorescence anisotropy binding measurements indicated that the preferred substrates for YicC were relatively small RNAs that contain some secondary structure. Larger RNAs or highly structured RNAs were less-than-optimal substrates. Similarly, RyhB RNA, a [~]90-nucleotide, iron-responsive RNA of E. coli, which has been described as a target of YicC binding and/or cleavage, was a poor YicC substrate in our assays. These results suggest that the native substrates for YicC-family members are very small RNAs or RNA fragments derived from larger RNAs.

Stress Granules (SGs) are cytoplasmic biomolecular condensates that form in response to a variety of stress conditions, though their function remains unclear. "Canonical" SGs - caused by stressors like sodium arsenite - are dynamic and cytoprotective, allowing cells to evade cell death during periods of stress. Ultraviolet (UV) irradiation is known to elicit a "non-canonical" SG subtype, lacking canonical SG components such as eukaryotic initiation factor 3 and polyadenylated mRNAs. The exact function of UV SGs, and the mechanisms driving their formation, remain unknown. Here we report the findings of a comparative analysis of UVA, UVB and UVC exposures on SG formation in three cell types: osteosarcoma (U2OS), keratinocytes (HaCaT), and mouse embryonic fibroblasts (MEF). We observed that SG formation in response to UV is highly cell type dependent. UVB and UVC induce robust SG formation in U2OS cells. However, only UVC exposure induced modest SG formation in MEFs, and none of the wavelengths caused SGs in HaCaT. While UVC-induced SGs in U2OS cells appear to be cell cycle dependent and specific to G1, UVB induced SG formation regardless of cell cycle stage. We tested the hypothesis that oxidative stress triggered by UV may be driving UV SG formation, and that keratin may buffer this effect, by overexpressing keratin in U2OS. Interestingly, we found that keratin and antioxidant treatment efficiently suppressed arsenite-induced SGs but had no effect on UV SGs. Our work confirms that UV SG formation is cell type specific and is not driven by oxidative stress.

Mycobacterium tuberculosis topoisomerase I (MtbTOP1) is essential for the viability of the causative agent of TB. There are still significant unanswered questions regarding the dynamic conformations during catalysis of relaxation of negatively supercoiled DNA by MtbTOP1. We aim to study the flexible hinge residues that control the dynamics of inter-domain rearrangements involved in the enzyme conformational changes that allow the opening-closing of the topoisomerase gate. We used the online server PACKMAN to predict possible hinges from the MtbTOP1 crystal structure. The predicted region "PRO506 to LEU526" at the border between domains D2 and D4 with a p-value <0.05 was then studied as a potential hinge. The highly conserved ARG516 from this region interacts with the DNA inside the protein toroidal cavity. This arginine maintains inter-domain interaction with GLU207 of D4 and ASP691 of D5 domains. After introducing alanine substitutions, we further studied the mutant topoisomerases in biochemical experiments. The results showed a significant loss in DNA relaxation activity without affecting DNA binding and cleavage after mutating GLU207 and ARG516, consistent with their role as hinge residues in domain rearrangements.